Cardiomyocytes derived from embryonic stem cells (ESCM) represent a novel and potentially life- saving treatment of heart disease. However, ESCM electrical behavior is more characteristic of the early embryonic, than the adult heart. ESCM display spontaneous electrical activity so that transplantation of these cells may lead to lethal arrhythmias. Preliminary results suggest that the spontaneous activity is related to a lack of a specific K ion conductance that normally increases during embryonic development. The Project will test approaches for producing ESCM that more closely duplicate normal adult heart cell function. Maturation will be evaluated with respect to electrophysiology, Ca signaling properties and ultra structure. In Aim 1 we will determine the time- dependent changes of ESCM, as characterized by their functional and structural properties. Whole- cell and single-channel K currents, T-type and L-type Ca currents, fast and slow Na currents, and global Ca transients and Ca sparks will be measured in individual ESCM. In Aim 2 we will introduce the K conductance into ESCM to transition the electrical activity towards the adult ventricular phenotype. We will test the hypothesis that changes in electrical activity mediated by this K conductance alter intracellular Ca signaling, and thereby the degree of ESCM maturation. In Aim 3 a three-dimensional environment of ESCM will be employed to test the relationship between functional expression of the K conductance and degree of maturation. In Aim 4 electrical coupling between co- cultured ESCM and native myocytes will be evaluated for normal electrical conduction and the absence of arrhythmias. The Project will provide a greater understanding of early cardiac development as recapitulated in the ES-derived myocytes, and the role of membrane potential signaling in promoting myocyte differentiation towards a mature ventricular phenotype. The long-term goal will be the production of terminally-differentiated myocytes displaying structural and functional characteristics of the adult ventricular myocardium which will be more useful in replacing myocardium damaged by disease. The results of the Project will bring us significantly closer to the realization of this promising treatment for cardiovascular disease, including ischemic heart disease and heart failure, in the adult and increasingly aged populations. PUBLIC HEALTH RELEVANCE: Heart disease is a leading cause of death in the U.S. This Project will provide new information on how embryonic stem cells can be used to produce mature replacement cells for damaged or diseased heart tissue.